1. Field of the Invention
In humans and mice, the membrane bound form of CD14 (mCD14) found on the cell surface of monocytes, macrophages and polymorphonuclear neutrophils (PMN) mediates the activation of these cells by lipopolysaccharide (LPS). Soluble CD14 (sCD14), present in the circulation, binds to LPS and blocks its binding to mCD14. In addition, sCD14, present in milk, is involved in sensitization of mammary duct epithelial cells and induction of an immune response. This invention relates to the cloning and expression of a form of recombinant bovine soluble CD14, identified here as rbosCD14, and its functional roles.
2. Description of the Relevant Art
Bovine coliform mastitis is an inflammation of the mammary gland caused by Gram-negative bacteria, where Escherichia coli is the most common pathogen. Mastitis is the most costly disease in the dairy industry, with economic losses of approximately two billion dollars annually in the United States. Mastitis results in decreased milk production, increased veterinary costs, and early culling or death of animals. Coliform mastitis is the most prevalent form of clinical mastitis, with infection by E. coli being the most frequent. About 80% of all intramammary infections by coliform bacteria will result in clinical mastitis, and 10% will result in peracute mastitis with a sudden onset of septic shock. Because coliforms are present in the cow's environment, they cannot be eradicated on a practical basis. Conventional herd management practices such as pre- and post-milking teat dipping and dry cow antibiotic therapy are unable to reduce the incidence of new infections. Coliform mastitis will exist as an animal health problem even in well managed herds. Conventional antibiotic treatment, extensive fluid supplementation, and metabolic support are not effective in relieving disease symptoms (1996. Current Concepts of Bovine Mastitis. National Mastitis Council, Madison, Wis.). Therefore, it is important to develop novel therapeutic regimens to control symptoms associated with acute coliform mastitis. Understanding the pathophysiological response of the mammary gland to coliform infections is critical in order to design such novel preventive and therapeutic regimens for clinical coliform mastitis.
It has been postulated that microbial products, often present in adjuvants, act on the innate immune system to elicit signals for activation of the adaptive immune system (Janeway, C. A. Jr. 1989. CSHSQB 54 (Pt 1): 1-13; Medzhitov et al. 1997. Curr. Opin. Immunol. 9: 4-9). Kinetic studies of experimental coliform mastitis induced by the intramammary injection of E. coli showed that inflammatory response will not be initiated until bacterial growth reaches a certain level (Shuster et al. 1995. Proc. Soc. Exp. Biol. Med. 210: 140-149; Shuster et al. 1996. Am J. Vet. Res. 57: 1569-1575). Uncontrolled bacterial growth results in a buildup in the concentration of microbial products that can be recognized by the host as a danger signal for the presence of a bacterial infection. It is conceivable that this signal is comprised of a conserved group of molecules across bacterial groups, and the host processes a sensitive machinery to detect this danger signal after it exceeds a certain threshold. Lipopolysaccharide (LPS) is one of the best characterized candidates of a danger signal because LPS is a component of the outer membrane of all Gram-negative bacteria and is released by actively growing, damaged, and dead bacteria (Petsch et al. 2000. J. Biotechnol. 76: 97-119). The toxicity of LPS is attributed to lipid A, a conserved domain of LPS.
CD14 is a receptor that binds to LPS and mediates the LPS-induced activation of host cells (Wright et al., supra). Two forms of CD14 exist. Membrane bound CD14 (mCD14) is present on the cell surface of monocytes, macrophages, and PMN, and mediates activation of those phagocytes by low concentrations of LPS in the presence of LPS-binding protein (LBP). Soluble CD14 (sCD14) is present in serum/plasma and urine of nephritic patients (Maliszewski et al. 1985. J. Immunol. 135: 1929-1936; Bazil et al. 1986. Eur. J. Immunol. 16: 1583-1589; Haziot et al. 1988. J. Immunol. 141: 547-552), and mediates activation of cells not bearing mCD14, including epithelial cells and endothelial cells (Arditi et al. 1993. Infect. Immun. 61: 3149-3156; Frey et al. 1992. J. Exp. Med. 176: 1665-1671; Pugin et al. 1993. Proc. Natl. Acad. Sci. USA 90: 2744-2748; Read et al. 1993. Proc. Natl. Acad. Sci. USA 90: 9887-9891). Macrophages are the predominant cell type in milk from uninfected bovine mammary glands. Bovine macrophages and PMN in milk express mCD14 on their cell surface (Paape et al. 1996. Am. J. Vet. Res. 57: 477-482). Labeta et al. (2000. J. Exp. Med. 191: 1807-1812) have reported the detection of sCD14 in human milk. Because bovine mammary epithelial cells do not express mCD14 as determined by PCR (data not shown), we have postulated that shedding of mCD14 from cell surfaces of milk macrophages and PMN is probably the major source of sCD14 in bovine milk. We too have detected sCD14 in human milk using anti-human CD14 mAb 60bca by Western blot; however, the concentration of sCD14 in bovine milk has not been heretofore determined due to the lack of a reliable ELISA for measuring bovine sCD14.
Binding of LPS to mCD14 on the surface of mCD14-bearing cells in the presence of LPS binding protein initiates the production of pro-inflammatory cytokines and mediators that are necessary for the host defense against infection by Gram-negative bacteria (Kurland et al. 1978. J. Exp. Med. 147: 952-957; Dentener et al., supra). For example, binding leads to an increase in the adhesion of PMN to a fibrinogen-coated surface (Wright, supra), translocation of nuclear factor κβ(NF-κβ, Morrison et al. 1979. Adv. Immunol. 28: 293-450), and the release of tumor necrosis factor-α (TNF-α) by monocytes and macrophages (Dentener, supra), all indicators of activation. However, overwhelming release of cytokines and pro-inflammatory mediators can be detrimental to the host (Bass et al. 1998. In: Phagocyte Function: A Guide for Research and Clinical Evaluation, J. P. Robinson and G. F. Babcock, Eds. Wiley-Liss, New York, pages 97-123). In severe conditions such as endotoxemia or septic shock, systemic responses induced by LPS can result in fever, hypertension, and organ injury (Bone, R. C. 1991. Chest 100: 802-808). Due to the lack of an effective treatment, mortality associated with Gram-negative septic shock in hospitals remains high at 25-30% (Glauser et al. 1991. Lancet 338: 732-739). The addition of sCD14 inhibits these activation effects of LPS on leukocytes in vitro by binding LPS and preventing its interaction with mCD14 (Maliszewski, C. R. 1991. Science 252: 1321-1322; Haziot et al. 1994. J. Immunol. 152: 5868-5876; Juan et al. 1995. J. Biol. Chem. 270: 1382-1387). The inhibitory effect of human sCD14 on LPS-induced activation of leukocytes protects mice from a lethal challenge of LPS (Haziot et al. 1995. Prog. Clin. Biol. Res. 392: 349-351). Therefore, sCD14 may be a potential therapeutic tool in controlling the acute inflammatory response caused by Gram-negative bacterial infections such as is seen in bovine coliform mastitis.
In addition, mammals process a CD14-dependent pathway to detect subpicomolar concentrations of LPS that activate host cells to mount an inflammatory response for clearance of bacteria (Dentener et al. 1993. J. Immunol. 150: 2885-2891; Ulevitchand et al. 1999. Curr. Opin. Immunol. 11: 19-22; Wright et al. 1990. Science 249: 1431-1433). Studies using LBP−/− deficient mice have shown that defects in CD14-dependent cellular responses to LPS protected mice from a lethal challenge with LPS, but prevented bacterial clearance after bacterial challenge in vivo (Jack et al. 1997. Nature 389: 742-745; Wurfel et al. 1997. J. Exp. Med. 186: 2051-2056). A delay in leukocyte recruitment after intramammary coliform infections leads to approximately 10 times more bacteria (Erskine et al. 1989. Am. J. Vet. Res. 50: 2093-2100). These studies emphasize the importance of a rapid and early inflammatory reaction in protecting the host from an overwhelming bacterial infection.
Previous studies have shown that human sCD14 forms a complex with LPS and mediates activation of cells not bearing mCD14 in the presence of low concentrations of LPS (Pugin et al., Frey et al., supra). Therefore, bovine soluble CD14-induced activation may contribute to udder swelling and changes in vascular and mammary epithelium permeability, which are the first two clinical signs observed after experimental coliform infections (Shuster et al. 1995, 1996, supra). Experiments to determine correlations among mammary gland sensitivity to LPS, milk somatic cell counts (MSCC), and the incidence of coliform mastitis after experimental exposure to E. coli facilitate the understanding of the initiation of inflammatory response in the bovine mammary gland making possible the designing of regimens for controlling coliform mastitis.
The production of a form of recombinant bovine soluble CD14, i.e., rbosCD14, by a baculovirus and insect cell expression system provides both a useful research tool for studying cattle immune responses to LPS and a potential therapeutic regimen for bovine diseases caused by Gram-negative bacteria, such as is the case with peracute coliform mastitis. As a bovine therapeutic, there is a role for a recombinant sCD14 molecule both as an inhibitor of the activation which occurs in an acute inflammatory response and as a sensitizer of bovine mammary ductal epithelial cells, thereby serving as an early signal for recruitment of leukocytes and activation of an immune response.